Salmonellosis continues to be a major world-wide health concern. Central to the pathogenicity of Salmonella enterica is its ability to engage host cells in an intimate two-way biochemical interaction. The centerpiece of this interaction is a type III protein secretion system encoded at centisome 63 of its chromosome. This system directs the translocation into the host cell of several bacterial effector proteins that stimulate a variety of cellular responses including actin cytoskeleton rearrangements, membrane ruffling, macropinocytosis, activation of transcription factors and, in some cell types such as macrophages, the stimulation of program cell death. These responses are critical for pathogenicity as they allow the bacteria to gain access to host cells, avoid host defense mechanisms and reach deeper tissues. In addition, they contribute to the establishment of the inflammatory diarrhea that most often ensues Salmonella infections by stimulating the production of pro-inflammatory cytokines. Work in our laboratory supported by this Grant has focused on the study of the cell biology of these Salmonella-host cell interactions. During the last funding period we established that central to these responses is the function of the small molecular weight GTP- binding protein CDC42. Through different downstream effectors that remain largely unidentified, this G protein orchestrates both actin cytoskeleton rearrangements resulting in bacterial entry, as well as the activation of transcription factors leading to cytokine production. Our understanding of this process has been helped greatly by the identification of the actual bacterial effectors that trigger these responses after their delivery through the type III secretion system. The proposed research project is aimed at gaining a better understanding of the cell biology of the cellular responses induced by Salmonella enterica as well as a more thorough understanding of the mechanism of action of the different bacterial effector proteins. More specifically we propose: 1) To define the role of different CDC42 downstream target effector proteins in the Salmonella-induced nuclear and cytoskeletal responses; 2) To investigate the function of the S. typhimurium effectors SipA, SopE, SopB, and SptP which are delivered into the host cell via the centisome 63 type III secretion system; and 3) To initiate studies to investigate the potential in-vivo role of signaling molecules during bacterial infection. These studies will advance the understanding of the cell biology of Salmonella enterica infections and facilitate the development of novel immunological and pharmacological strategies to prevent diseases caused by all Salmonella enterica serovars including S. typhi.